Objectives To determine the beta-lactam exposure associated with positive clinical outcomes for Gram-negative blood stream infection (BSI) in critically ill patients. Patients and methods Pooled data of critically ill patients with mono-microbial Gram-negative BSI treated with beta-lactams were collected from two databases. Free minimum concentrations (fC(min)) of aztreonam, cefepime, ceftazidime, ceftriaxone, piperacillin (co-administered with tazobactam) and meropenem were interpreted in relation to the measured MIC for targeted bacteria (fC(min)/MIC). A positive clinical outcome was defined as completion of the treatment course or de-escalation, without other change of antibiotic therapy, and with no additional antibiotics commenced within 48h of cessation. Drug exposure breakpoints associated with positive clinical outcome were determined by classification and regression tree (CART) analysis. Results Data from 98 patients were included. Meropenem (46.9%) and piperacillin/tazobactam (36.7%) were the most commonly prescribed antibiotics. The most common pathogens were Escherichia coli (28.6%), Pseudomonas aeruginosa (19.4%) and Klebsiella pneumoniae (13.3%). In all patients, 87.8% and 71.4% achieved fC(min)/MIC >= 1 and fC(min)/MIC >5, respectively. Seventy-eight patients (79.6%) achieved positive clinical outcome. Two drug exposure breakpoints were identified: fC(min)/MIC >1.3 for all beta-lactams (predicted difference in positive outcome 84.5% versus 15.5%, P<0.05) and fC(min)/MIC >4.95 for meropenem, aztreonam or ceftriaxone (predicted difference in positive outcome 97.7% versus 2.3%, P<0.05). Conclusions A beta-lactam fC(min)/MIC >1.3 was a significant predictor of a positive clinical outcome in critically ill patients with Gram-negative BSI and could be considered an antibiotic dosing target.